A versatile gene trap to visualize and interrogate the function of the vertebrate proteome
- Creators
- Trihn, Le A.
- Hochgreb, Tatiana
- Graham, Matthew
- Wu, David
- Ruf-Zamojski, Frederique
- Jayasena, Chathurani S.
- Saxena, Ankur
- Hawk, Rasheeda
- Gonzales-Serricchio, Aidyl
- Dixson, Alana
- Chow, Elly
- Gonzales, Constanza
- Leung, Ho-Yin
- Solomon, Ilana
- Megason, Sean G.
- Fraser, Scott E.
- Bronner-Fraser, Marianne
Abstract
We report a multifunctional gene-trapping approach, which generates full-length Citrine fusions with endogenous proteins and conditional mutants from a single integration event of the FlipTrap vector. We identified 170 FlipTrap zebrafish lines with diverse tissue-specific expression patterns and distinct subcellular localizations of fusion proteins generated by the integration of an internal citrine exon. Cre-mediated conditional mutagenesis is enabled by heterotypic lox sites that delete Citrine and "flip" in its place mCherry with a polyadenylation signal, resulting in a truncated fusion protein. Inducing recombination with Cerulean-Cre results in fusion proteins that often mislocalize, exhibit mutant phenotypes, and dramatically knock down wild-type transcript levels. FRT sites in the vector enable targeted genetic manipulation of the trapped loci in the presence of Flp recombinase. Thus, the FlipTrap captures the functional proteome, enabling the visualization of full-length fluorescent fusion proteins and interrogation of function by conditional mutagenesis and targeted genetic manipulation.
Additional Information
© 2011 by Cold Spring Harbor Laboratory Press. The Authors acknowledge that six months after the full-issue publication date, the Article will be distributed under a Creative Commons CC-BY-NC License (Attribution-NonCommercial 4.0 International License, http://creativecommons.org/licenses/by-nc/4.0/). Received July 7, 2011; revised version accepted September 16, 2011. We thank P. Pantazis for comments on the manuscript, and members of the Fraser laboratory for helpful discussions. We are grateful to LeighAnn Fletcher for fish care. The T2KXIGd-in and pMDS-eGFP plasmids were provided by K. Kawakami and S. Parinov, respectively. This work was supported by NHGRI Center of Excellence in Genomic Science grant P50HG004071.Attached Files
Published - Genes_Dev.-2011-Trinh-2306-20.pdf
Published - Trinh2011p16329Genes_Dev.pdf
Supplemental Material - Binder2.pdf
Supplemental Material - ResourcePaperSI_text.pdf
Files
Additional details
- PMCID
- PMC3219234
- Eprint ID
- 27984
- Resolver ID
- CaltechAUTHORS:20111129-075143013
- NIH
- P50HG004071
- Created
-
2011-11-29Created from EPrint's datestamp field
- Updated
-
2021-11-09Created from EPrint's last_modified field